Introduction: Sexually transmitted infections (STIs) are a major public health concern worldwide, affecting quality of life and causing serious morbidity and mortality. Neisseria gonorrhoea is challenging to diagnose by culture due to its fastidious nature. A rapid, highly sensitive PCR assay is urgently needed for routine N. gonorrhoea detection to prevent widespread infection and reproductive complications. The aim of this study was to evaluate the sensitivity of an in-house PCR assay specifically for the detection of N. gonorrhoeae.
Methods: For the design of the in-house PCR, the 16S rRNA of a N. gonorrhoeae isolate was accessed from GenBank (AJ247239.2). Using a primer design tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/), the primers were selected and used in a qPCR SYBR green assay. The in-house 16S rRNA assay was compared to the commercially available TaqMan assay which uses primers and probes specific for N. gonorrhoeae. The 16SrRNA primers were tested for cross reactivity towards six Neisseria strains. The DNA from the following Neisseria isolates: N. elongate, N. cinera, N. weaver and N. sicca were tested.
Results/findings: The analysis showed successful 16S rRNA amplification in all clinical N. gonorrhoea isolates. Positive amplification matched in-house and TaqMan results, displaying strong correlation. Cross-reactivity analysis confirmed assay specificity for N. gonorrhoeae detection, excluding non-gonococcal Neisseria strains.
Conclusion: This study showed that the in-house PCR had successfully detected N. gonorrhoeae. The in-house assay showed a good correlation with a commercially available assay. This assay needs to be validated using different clinical samples to show its promise as a diagnostic test.
Keywords: Neisseria gonorrhoea, Cross-reactivity, TaqMan assay, In-house PCR assay